Benzo[a]pyrene Flow-Hysteresis Immuno-Time-Resolved Fluorescence Rapid Test Kit and Its Application

Abstract

The present invention relates to a benzo[a]pyrene flow-lag immunoassay time-resolved fluorescence rapid test kit and its application. The kit comprises a fluorescent test strip and a sample reaction cup containing a europium-labeled fluorescent probe, wherein the europium-labeled fluorescent probe is a freeze-dried europium-labeled anti-benzo[a]pyrene monoclonal antibody. The fluorescent test strip comprises a backing plate, one side of which is sequentially adhered with an absorbent pad, a test pad, and a sample pad, with adjacent pads overlapping at their junctions. The test pad is based on a nitrocellulose membrane and has a quality control line and a test line arranged horizontally from top to bottom. The quality control line and the test line are coated with a goat anti-mouse secondary antibody and a benzo[a]pyrene detection antigen, respectively. The kit is simple to operate and can accurately and rapidly determine the benzo[a]pyrene content in a sample.

Description

Technical Field

[0001] The present invention belongs to the field of mycotoxin detection, and in particular relates to a benzo[a]pyrene flow lag time resolved fluorescence rapid detection kit and application thereof. Background Art

[0002] Benzo[a]pyrene (B[a]P) is a chronic teratogenic and carcinogenic substance. Long-term exposure to air containing B[a]P can cause chronic poisoning. It primarily enters the human body through food or water, is absorbed through the intestines, and circulates throughout the body, accumulating in the mammary glands and adipose tissue. Benzo[a]pyrene is a strong irritant to the skin and eyes and is a mutagen and carcinogen that can cause human diseases. Animal studies have shown that B[a]P ingested directly can enter the fetus through the placenta, causing varying degrees of toxicity and carcinogenicity. Benzo[a]pyrene is excreted in feces after passing through detoxification organs such as the liver and kidneys. Benzo[a]pyrene excrement can be applied to the land as fertilizer, contaminating crops and ultimately affecting human health. Benzo[a]pyrene is more toxic than aflatoxin and is not only the most toxic of the PAHs but also one of the most abundant, comprising approximately one-fifth of all carcinogenic PAHs in the environment. Animal experimental studies have shown that polycyclic aromatic hydrocarbons, especially 3,4-benzo[a]pyrene, are related to lung cancer in animals and humans and are the cause of lung cancer.

[0003] With the continuous improvement of living standards, people have higher and higher requirements for food quality and safety. In order to improve the quality and safety of food in my country and meet people's safe consumption needs to a greater extent, accurate and efficient detection technology for benzo[a]pyrene in food is urgently needed.

[0004] Currently, the main methods for detecting benzo[a]pyrene are instrumental analysis and immunoassays. Instrumental analysis methods, such as high-performance liquid chromatography and liquid chromatography-mass spectrometry, offer high sensitivity and accuracy. However, these methods are expensive, require complex and time-consuming sample pretreatment, and require a high laboratory environment, making rapid detection difficult. Immunoassays, based on the specific recognition and reversible binding reaction between antigens and antibodies, offer high selectivity and sensitivity. Compared to instrumental analysis, they significantly simplify and shorten sample processing time, saving testing costs.

[0005] Due to the increasing trend of benzo[a]pyrene contamination in cereals reported in numerous publications in recent years, there is an urgent need for rapid on-site detection technologies for benzo[a]pyrene contamination to ensure food safety in my country. Currently, there are no immunofluorescence time-resolved detection kits for benzo[a]pyrene detection on the market. Summary of the Invention

[0006] The present invention aims to provide a benzo[a]pyrene flow-lag immunoassay time-resolved fluorescence rapid detection kit and its application, which is simple to operate and can accurately and quickly determine the benzo[a]pyrene content in a sample.

[0007] The technical solution adopted in the present invention is as follows:

[0008] A benzo[a]pyrene flow-lag immunoassay time-resolved fluorescence rapid test kit comprises a fluorescent test strip and a sample reaction cup containing a europium-labeled fluorescent probe, wherein the europium-labeled fluorescent probe is a freeze-dried europium-labeled anti-benzo[a]pyrene monoclonal antibody. The fluorescent test strip comprises a backing plate, one side of which is sequentially adhered with a water-absorbing pad, a detection pad, and a sample pad from top to bottom, with adjacent pads overlapping and connected at the joint. The detection pad is based on a nitrocellulose membrane and has a quality control line and a detection line arranged horizontally from top to bottom. The quality control line and the detection line are respectively coated with a goat anti-mouse secondary antibody and a benzo[a]pyrene detection antigen. The anti-benzo[a]pyrene monoclonal antibody is obtained by secretion of a hybridoma cell line BBBE1H1. The hybridoma cell line BBBE1H1 was deposited in the China Center for Type Culture Collection (CCTCC) on April 3, 2018, at Wuhan University, Wuhan, China, with a deposit number of CCTCC. NO:C201882.

[0009] According to the above scheme, the europium-labeled anti-benzo[a]pyrene monoclonal antibody lyophilized product is formed by coupling the surface-modified carboxyl group with the amino terminus of the anti-benzo[a]pyrene monoclonal antibody. The specific preparation method is as follows: the europium labeling reagent is added to the boric acid buffer and sonicated, and then the EDC solution is added, oscillated and mixed, centrifuged, the supernatant is removed, the boric acid buffer is added for re-dissolution, oscillated and mixed, the anti-benzo[a]pyrene monoclonal antibody is added, the reaction is shaken, the supernatant is removed by centrifugation, the solution is sealed, and the solution is packaged and lyophilized.

[0010] According to the above scheme, the europium labeling reagent is europium oxide latex fluorescent microspheres.

[0011] According to the above scheme, the EDC solution is activated by oscillation for 15-30 minutes, the centrifugal speed is 10000-15000 rpm, and the reaction is shaken for 2-4 hours; the blocking is to add borate buffer containing 0.5-1% BSA to the precipitate after centrifugation to re-dissolve and block the excess binding sites on the surface of the europium labeling reagent.

[0012] According to the above scheme, the benzo[a]pyrene detection antigen is BAP-OVA.

[0013] According to the above scheme, the coating amount of benzo[a]pyrene detection antigen required for each centimeter of detection line on the detection pad of the fluorescent test strip is 480-1000 ng; the coating amount of goat anti-mouse polyclonal antibody required for each centimeter of quality control line is 100-900 ng; and the content of fluorescent probe in the sample reaction cup is 0.2-1.0 μg.

[0014] According to the above scheme, the para[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit further includes a sample release solution and a sample release solution pipette, wherein the sample release solution is: a PBS solution (v / v) containing 1.0% to 1.5% sucrose (w / w), 0.05% to 3.0% BSA (w / w) and 0.01% to 0.30% Tween-20;

[0015] The fluorescent test strip is obtained by the following method:

[0016] (1) Cut the absorbent paper into absorbent pads of the required size;

[0017] (2) Preparation of test pad:

[0018] The benzo[a]pyrene detection antigen is prepared into an antigen coating solution, which is then horizontally coated on a nitrocellulose membrane by streaking to obtain a test line; the goat anti-mouse secondary antibody is prepared into a coating solution, which is then horizontally coated on a nitrocellulose membrane by streaking to obtain a quality control line;

[0019] (3) Preparation of sample pad:

[0020] Place the glass fiber membrane in the blocking solution and soak it completely. Take it out and dry it at 37-40℃ overnight to obtain the required sample pad for later use.

[0021] (4) Assembly of fluorescent test strips:

[0022] The absorbent pad, detection pad and sample pad are sequentially pasted on one side of the liner. Adjacent pads are overlapped at the joints to obtain a fluorescent test strip, with the overlapping length between them being 1 to 2 mm.

[0023] According to the above scheme, the sample pad in the test strip is 13mm-15mm long, the test pad is 25mm-28mm long, and the absorbent pad is 17mm-20mm long. The distance between the test line and the top edge of the nitrocellulose membrane is 10-18mm, and the distance between the quality control line and the test line is 2-6mm.

[0024] According to the above scheme, the test strip is 3 to 5 mm wide and 45 to 65 mm long, and is placed in a test strip tube in a desiccant and stored at 4°C.

[0025] According to the above scheme, the sample pad blocking solution is: 1g ovalbumin, 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, added with water to make up to 100mL;

[0026] The application of the above-mentioned benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit in the detection of benzo[a]pyrene content is as follows: a sample reaction cup is placed in a 37°C incubator, a sample solution to be tested is added to the sample reaction cup, pipetted and mixed, and then a fluorescent test strip is inserted. After a reaction time of 6 to 10 minutes, the test is performed using a time-resolved fluorescence tester to obtain the ratio of the fluorescence intensity value of the test line (T) on the fluorescent test strip to the fluorescence intensity value of the quality control line (C); based on the pre-obtained relationship curve between the ratio of the fluorescence intensity of the test line of the fluorescent test strip to the fluorescence intensity of the quality control line (T / C) and the benzo[a]pyrene concentration, the benzo[a]pyrene content in the sample solution to be tested is obtained.

[0027] According to the above scheme, the sample to be tested is edible vegetable oil, and the pretreatment steps of the sample to be tested are: ultrasonic extraction of the edible vegetable oil with n-hexane, taking the supernatant, diluting it 8-12 times with water, then adding immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with a sample release solution to obtain the supernatant, which is the sample solution to be tested.

[0028] The immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies comprise magnetic beads and anti-benzo[a]pyrene monoclonal antibodies coupled via COOH groups on the magnetic beads and amino groups on the antibodies. The mass ratio of the anti-benzo[a]pyrene monoclonal antibodies to the magnetic beads in the immunomagnetic beads is 2:1 to 1:5.

[0029] According to the above scheme, the detection solution is PBS containing 0.5wt% Tween 20, pH 7.4.

[0030] According to the above scheme, the relationship curve between the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the quality control line (T / C) and the concentration of benzo[a]pyrene is obtained by the following method:

[0031] (1) preparing a series of benzo[a]pyrene standard solutions with a concentration gradient;

[0032] (2) Add appropriate amounts of the above-mentioned benzo[a]pyrene standard solutions of each concentration to the sample reaction bottle, mix well, insert the fluorescent test strips, react at 37°C for 6 minutes, and use a time-resolved fluorescence immunoassay to obtain the time-resolved fluorescence intensity values of the test line (T) and the quality control line (C) on each fluorescent test strip, thereby obtaining the ratio of the fluorescence intensity of the test line of each fluorescent test strip to the fluorescence intensity of the quality control line (T / C);

[0033] (3) The relationship curve between the ratio of the fluorescence intensity of the test line of the fluorescent test strip to the fluorescence intensity of the quality control line (T / C) and the concentration of benzo[a]pyrene was obtained by fitting.

[0034] In this study, a benzo[a]pyrene monoclonal antibody was coupled with a rare earth element europium labeling reagent to develop a highly sensitive benzo[a]pyrene time-resolved fluorescence immunochromatographic test strip. A time-resolved fluorescence immunochromatographic method was also established. This method has the advantages of high sensitivity, stable properties, avoidance of fluorescence background interference, and short detection time (generally only 6-8 minutes). It is very suitable for the development of rapid pesticide residue detection methods and can achieve rapid and sensitive detection of benzo[a]pyrene.

[0035] Beneficial effects of the present invention:

[0036] The present invention provides a benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit that can accurately and rapidly determine benzo[a]pyrene content. The sample detection limit for benzo[a]pyrene is 0.5 ng / mL (blank sample tested 11 times, with the SD calculated, resulting in a detection limit of 3 SD). The test strip was applied to actual sample testing, and the test results compared with HPLC methods showed a recovery rate between 90.3% and 110.2%, with a correlation coefficient of 0.987 (R²), indicating its applicability to actual sample testing of benzo[a]pyrene. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the structure of the fluorescent test strip in the benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit provided by the present invention. In the figure: 1 sample pad, 2 test line, 3 quality control line, 4 absorbent pad. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific implementation cases.

[0039] Example 1: Preparation of anti-benzo[a]pyrene monoclonal antibodies

[0040] The anti-benzo[a]pyrene monoclonal antibody is secreted by the BBBE1H1 hybridoma cell line with a deposit number of CCTCC NO: C201882, and is prepared by:

[0041] The anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1 was injected into BALB / c mice that had been pre-treated with Freund's incomplete adjuvant. The ascites of the mice were collected, and the antibodies were purified using the octanoic acid-ammonium sulfate method. The specific operation was as follows: the mouse ascites was filtered through double filter paper, centrifuged at 12,000 rpm at 4°C for more than 15 minutes, and the supernatant was aspirated. The resulting ascites supernatant was mixed with 4 volumes of acetate buffer, and octanoic acid was slowly added with stirring. The volume of octanoic acid required per milliliter of ascites was 30-35 μL. The mixture was mixed at room temperature for 30-60 minutes and allowed to stand at 4°C for more than 2 hours. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double filter paper, add 1 / 10 volume of filtrate with a molar concentration of 0.1mol / L and a pH of 7.4 phosphate buffer, adjust the pH of the mixture to 7.4 with 2mol / L sodium hydroxide solution, slowly add ammonium sulfate in an ice bath to a final concentration of ammonium sulfate of 0.277g / mL, let it stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, and resuspend the obtained precipitate with 1 / 10 volume of original ascites water with a molar concentration of 0.01mol / L and pH of 7.4 phosphate buffer, put it into a dialysis bag, dialyze it with 0.01mol / L PBS for two days, and then dialyze it with PB for two days. Remove the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then freeze-dry it in a freeze dryer. Collecting the lyophilized powder is the purified anti-benzo[a]pyrene monoclonal antibody;

[0042] The acetate buffer solution is prepared by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to make the volume to 100 mL; the 0.01 mol / L phosphate buffer solution is prepared by adding water to 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to make the volume to 100 mL; the 0.1 mol / L phosphate buffer solution is prepared by adding water to 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 0.2 g of potassium dihydrogen phosphate to make the volume to 100 mL.

[0043] The subtype of the anti-benzo[a]pyrene monoclonal antibody secreted by the hybridoma cell line BBBE1H1 was identified as IgG1 using a commercially available subtype identification kit.

[0044] The antibody titer purified from mouse ascites was measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) to be 1.2×10 5 , that is, the antibody dilution is 1.2×10 5The result of the solution test was positive when the concentration of the solution was doubled. The IC50 of its sensitivity to benzo[a]pyrene was determined by conventional indirect competitive ELISA to be 0.013 ng / mL. The specificity of the antibody can be evaluated by the cross-reaction rate. The indirect competitive ELISA method was used to determine the BBBE1H1 monoclonal antibody. A series of standard solutions of BaP, benzo[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene, and pyrene were prepared and added to the enzyme-labeled plate together with an equal volume of antibody. The solution was incubated at 37°C for 1 hour. The other steps were the same as the indirect competitive ELISA method. The competition inhibition curve was drawn with the concentration of the above-mentioned standard as the horizontal axis and the OD value B / B0 at 450 nm measured by the enzyme reader as the vertical axis. The IC 50 The cross-reaction rate is determined by the ratio of the values. The calculation formula is as follows:

[0045] CR%=(IC 50 BaP / IC 50 other analogs) × 100.

[0046] The cross-reactivity of the BBBE1H1 monoclonal antibody provided by the present invention with other structural analogs, benz[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene, and pyrene, is less than 15%, and some are as low as less than 1%.

[0047] Detailed results are shown in Table 1:

[0048] Table 1. Cross-reactivity of BBBE1H1 with other structural analogs

[0049]

[0050]

[0051] Affinity determination of BBBE1H1 using indirect non-competitive ELISA:

[0052] The ELISA plate was coated with BaP-OVA at concentrations of 2.0, 1.0, 0.5, and 0.25 μg / mL, 100 μL / well, at 37°C for 2 h. After blocking with blocking solution for 1 h, the antibody diluted with PBS (dilution factor 1:2) was added to the ELISA plate. The remaining steps were the same as those of the indirect non-competitive ELISA method. 450The value is the vertical axis, and the logarithm of the antibody concentration (mol / L) is the horizontal axis. Draw 4 S-shaped curves with 4 concentrations. Find the maximum OD value at the top of each S curve, i.e. ODmax, and find the antibody concentration corresponding to 50% ODmax value of each curve. Pair any two of the 4 concentrations together and calculate the affinity constant of the antibody according to the formula Ka=(n-1) / 2(n[Ab']t-[Ab]t), where [Ab']t and [Ab]t are the antibody concentrations corresponding to the two 50% maximum OD values in each group, and n is the multiple of the coating antigen concentration in each group (including three ratios of 1:2, 1:4, and 1:8). A total of 6 Ka values are obtained. The average of the six Ka values obtained shows that the affinity of the anti-benzo[a]pyrene mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method can reach 1.6×10 9 L / moL.

[0053] Example 2 Screening of hybridoma cell line BBBE1H1

[0054] 1. Animal immunization

[0055] Six-week-old female BALB / c mice were immunized with laboratory-prepared complete benzo[a]pyrene antigen (BaP-BSA). For the first immunization, the complete benzo[a]pyrene antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at five points on the back of the mouse's neck. The second immunization was performed 21 days later with an equal volume of the complete benzo[a]pyrene antigen emulsified with Freund's incomplete adjuvant and injected intraperitoneally. The third immunization was performed two weeks after the second, using the same immunization method. The fourth immunization was performed three weeks after the third, also using the same intraperitoneal injection. The same dose of 100 μg was administered per mouse for all four immunizations. Eight to ten days after each of the first three immunizations, blood was collected by tail-clip, and serum was isolated and assayed for serum titer using an indirect ELISA. Eight days after the third immunization, blood was collected by tail-clip, and mice with sera exhibiting relatively high titers and sensitivity were selected for a final booster immunization, using half the dose of the previous immunization.

[0056] 2. Cell Fusion

[0057] Three days after the booster immunization, cell fusion was performed using 50% polyethylene glycol (PEG) (molecular weight 1450) as a fusion agent according to conventional methods. The following steps were performed: mice were sacrificed by cervical dislocation under sterile conditions, the spleens were removed and crushed with a homogenizer, and splenocytes were separated using a filter. The cells were mixed with murine myeloma SP2 / 0 cells at a cell population ratio of 5:1-10:1, centrifuged at 1000 rpm for 5 minutes, and resuspended in RPMI-1640 basal medium. The mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. One mL of 50% PEG was added, and the mixture was incubated for 1 minute. Once adhered, cells were resuspended in 20 mL of RPMI-1640 basal medium, centrifuged, and the supernatant discarded. The fused cells at the bottom of the tube were resuspended in 20 mL of complete cell culture medium containing 1% HAT. The suspended cells were added to 80 mL of semi-solid culture medium, mixed thoroughly, and plated into a 6-well cell culture plate at a rate of 1-2 mL / well. The plates were then incubated in a 37°C CO2 incubator. The complete cell culture medium containing 1% HAT contains 20% (volume percentage) fetal bovine serum, 75% (volume percentage) RPMI-1640 basal culture medium, 1% (weight percentage) L-glutamine, 1% (volume percentage) HEPES, 1% (volume percentage) double antibody (10,000 units per milliliter penicillin and 10,000 micrograms per milliliter streptomycin), 1% (volume percentage) growth factor (clone easy), and 1% (weight percentage) hypoxanthine-aminopterin-thymidine (HAT) and methylcellulose, which were purchased from Sigma-Aldrich.

[0058] Cell line screening and cloning

[0059] After 1-2 weeks of cell fusion, when cell colonies grow to be visible to the naked eye, clones are picked out from the culture medium with a micropipette and transferred to a 96-well cell culture plate using HAT liquid for culture. When the cells grow to 2 / 3 of the bottom of the well, the culture supernatant is aspirated for detection. A two-step screening method is used. The first step uses the indirect ELISA method to screen positive wells that are resistant to benzo[a]pyrene but not to the carrier protein BSA. The second step uses the indirect competitive ELISA method to detect the positive wells screened in the first step, using benzo[a]pyrene as the competitor, and selecting wells with higher absorbance and sensitivity (higher absorbance refers to wells with zero competitor, i.e., positive control wells, with higher final measured values; higher sensitivity refers to the competitor concentration at which the inhibition rate is 50%, also known as IC). 50 The hybridoma cell line BBBE1H1 was obtained by subcloning using the limiting dilution method and testing using the same two-step method after subcloning 4-5 times. This hybridoma cell line was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on April 3, 2018, under the CCTCC accession number C201882.

[0060] Example 3 Determination of the variable region sequence of the anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1.

[0061] (1) Extraction of total RNA: Total RNA from hybridoma cell line BBBE1H1 was extracted using the total RNA extraction kit from Tiangen Company according to the instructions;

[0062] (2) Synthesize cDNA: Use the total RNA obtained in step 1 as a template and oligo(dT)15 as a primer. TM Reverse transcription was performed using the instructions of the 2II reverse transcriptase to synthesize the first-strand cDNA; the primer oligo(dT)15 was purchased from Invitrogen;

[0063] (3) PCR cloning of variable region genes: Primers were designed based on the conserved sites of mouse antibody gene sequences in GENBANK, and cDNA was used as a template to amplify the variable region genes of the antibody heavy and light chains. The PCR program was as follows: 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 1 minute, with 30 cycles of amplification and a final extension at 72°C for 10 minutes. The PCR products were separated by 1% (weight percent) agarose gel electrophoresis, and the DNA fragments were purified and recovered using a kit. The DNA fragments were ligated into the vector pMD18-T and transformed into Escherichia coli DH5α competent cells. Positive clones were picked and sent to Suzhou Hongxun Biotechnology Co., Ltd. for sequencing. The sequences of the primers are: heavy chain variable region primers are 5'-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCAGTG GAT AGA CAG ATG GGG G-3' (28mer), where S, M, R and W are merged bases, M = A / C, R = A / G, S = G / C, W = A / T; light chain variable region primers are 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24mer).

[0064] The resulting gene sequence results showed that the heavy chain variable region encoding gene sequence was 360 bp long, as shown in SEQ ID NO: 1. Based on the obtained gene sequence, it was deduced that the heavy chain variable region encoded by this gene sequence consists of 120 amino acids, as shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence was 321 bp long, as shown in SEQ ID NO: 2. Based on the obtained gene sequence, it was deduced that the light chain variable region encoded by this gene sequence consists of 107 amino acids, as shown in SEQ ID NO: 4.

[0065] Example 4: Benzo[a]pyrene Immunoassay Time-Resolved Fluorescence Rapid Assay Kit and Its Application

[0066] A benzo[a]pyrene flow-lag immunoassay kit for time-resolved fluorescence rapid detection includes a fluorescent test strip, a sample reaction bottle (containing a freeze-dried europium-labeled anti-benzo[a]pyrene monoclonal antibody), a sample diluent, and a sample diluent pipette. The fluorescent test strip consists of cardboard, with an absorbent pad, a test pad, and a sample pad adhered to one side of the cardboard in order from top to bottom. Adjacent pads overlap at the joints, with the overlap length being 1-2 mm. The absorbent pad is 15 mm long and 4 mm wide; the test pad is 25 mm long and 4 mm wide; and the sample pad is 13 mm long and 4 mm wide. The test pad is based on a nitrocellulose membrane, with a quality control line and a test line arranged horizontally from top to bottom on the nitrocellulose membrane. The quality control line is coated with a goat anti-mouse secondary antibody, and the test line is coated with a benzo[a]pyrene detection antigen. The distance between the test line and the top edge of the nitrocellulose membrane is 15 mm, and the distance between the quality control line and the test line is 5 mm.

[0067] Obtaining the fluorescent test strips:

[0068] (1) Preparation of absorbent pad

[0069] Cut the absorbent paper into 15mm long and 4mm wide specifications to make the absorbent pad;

[0070] (2) Preparation of detection pad

[0071] Test line coating:

[0072] Benzo[a]pyrene detection antigen (BAP-BSA) was prepared in coating buffer at a concentration of 0.8 mg / mL. The coating solution was applied horizontally to the nitrocellulose membrane using a line spray method at a distance of 15 mm from the top edge of the membrane to form a test line. The coating amount of BAP-BSA required per cm of the test line was 480 ng. The membrane was then dried at 37°C for 30 minutes.

[0073] The coating buffer is: 0.1 g bovine serum albumin, 0.002 g sodium azide, 0.08 g sodium chloride, 0.029 g disodium hydrogen phosphate dodecahydrate, 0.002 g potassium chloride, 0.002 g potassium dihydrogen phosphate, and water is added to make up to 10 mL;

[0074] Coating of quality control line:

[0075] Prepare a coating solution of 0.25 mg / mL of goat anti-mouse secondary antibody in coating buffer. Line-spray the solution onto a nitrocellulose membrane at a distance of 5 mm from the test line to create a control line. The coating amount of goat anti-mouse secondary antibody required per cm of the control line is 100 ng. Dry the membrane at 37°C for 1 hour.

[0076] The coating buffer is prepared by adding water to 10 mL of 0.1 g bovine serum albumin, 0.002 g sodium azide, 0.08 g sodium chloride, 0.029 g disodium hydrogen phosphate dodecahydrate, 0.002 g potassium chloride, and 0.002 g potassium dihydrogen phosphate;

[0077] (3) Preparation of sample pad:

[0078] Cut the glass fiber membrane into 13 mm long and 4 mm wide specifications, soak it in the blocking solution, take it out, dry it at 37°C for 6 hours to obtain the sample pad, and then store it in a desiccator at room temperature;

[0079] The blocking solution is prepared by adding water to 100 mL of 1 g ovalbumin, 2 g sucrose, 0.02 g sodium azide, 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, and 0.02 g potassium dihydrogen phosphate;

[0080] (4) Assembly of fluorescent test strips:

[0081] On one side of the cardboard, from top to bottom, stick the absorbent pad, detection pad, fluorescent labeled antibody reaction pad and sample pad in sequence. The adjacent pads are overlapped at the joints with an overlap length of 2 mm to obtain the fluorescent test strip (see Figure 1 ).

[0082] The europium labeling reagent, europium oxide latex, was prepared as follows: 800 μL of 0.2 mol / L pH 8.18 borate buffer was added to 200 μL of europium oxide latex (polystyrene fluorescent microspheres, particle size 200 nm, solids content 1%). The mixture was sonicated for 1 minute, and 40 μL of 15 mg / mL EDC solution was added. The mixture was vortexed for 15 minutes, followed by centrifugation (12,000 rpm, 10°C, 10 minutes). The supernatant was removed (to remove EDC and other substances), and 1 mL of borate buffer was added for reconstitution and vortexed. 10 ng of benzo[a]pyrene monoclonal antibody was added, and the mixture was shaken at 250 rpm and 4°C for 2 hours. The mixture was then removed and centrifuged (12,000 rpm, 10°C, 10 minutes). The supernatant was removed (to remove unbound antibody and BSA), and 1 mL of borate buffer containing 0.5% BSA was added for reconstitution. The mixture was vortexed and sonicated for 1 minute. The reaction was then shaken at 250 rpm and 4°C for 1 hour. After successful coupling, the mixture was aliquoted and lyophilized. The sample reaction vial containing the europium-labeled anti-benzo[a]pyrene monoclonal antibody lyophilized powder was obtained by placing 0.25 μg of the europium-labeled anti-benzo[a]pyrene monoclonal antibody in a 3 mL snap-top vial. After drying using a conventional freeze-vacuum drying method, the europium-labeled anti-benzo[a]pyrene monoclonal antibody lyophilized powder was obtained and stored at 4°C for future use.

[0083] Example 5: Preparation of the Benzo[a]pyrene Immunomagnetic Beads for Sample Pretreatment:

[0084] Preparation of Benzo[a]pyrene Immunomagnetic Beads:

[0085] a. Washing: Weigh 2.5 mg of carboxyl-modified magnetic beads into a 15 mL centrifuge tube, add 2 mL of washing solution and wash twice, place on a magnetic stand for magnetic separation, discard the supernatant, then wash twice with coupling buffer, magnetically separate, and discard the supernatant.

[0086] b Coupling: Add 4 mL of coupling buffer to the washed magnetic beads, add 2.5 mg of anti-benzo[a]pyrene monoclonal antibody, place on a shaker at 8°C, 200 rpm overnight for coupling. After the reaction is complete, place on a magnetic stand for magnetic separation.

[0087] c Blocking: Add Tris-HCl buffer to the coupled magnetic beads and wash twice, then add 4 mL of Tris-HCl blocking solution and react in a shaker at 8°C and 200 rpm for 2 h.

[0088] d. Storage: After the blocking reaction is complete, perform magnetic separation, discard the supernatant, dilute to 5 mL with 0.01% NaN3 in PBS, and store in a refrigerator at 4°C until use. This will be used for subsequent sample pretreatment.

[0089] Example 6: Application of the above-mentioned flow-delayed immunoassay time-resolved fluorescence rapid test kit in the detection of benzo[a]pyrene in vegetable oils:

[0090] Establishment of the relationship curve between the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the quality control line (T / C) and the concentration of benzo[a]pyrene:

[0091] (1) The vegetable oil samples that tested negative for benzo[a]pyrene by high performance liquid chromatography (HPLC) were pretreated and spiked with benzo[a]pyrene to concentrations of 200, 66.6, 22.2, 7.4, 2.4, 0.8, 0.2, and 0.09 ng / mL.

[0092] Sample pretreatment: Accurately weigh 1g of soybean oil in a 10mL plastic centrifuge tube as a reaction vessel and vortex mix for 1 minute. Add 5mL of n-hexane solution and sonicate for 10 minutes. Then add another 5mL of n-hexane solution and sonicate for 10 minutes. Take 1mL of the extract and dilute to 10mL with 9mL of ddH2O. Vortex for 2 minutes. Add immunomagnetic beads conjugated with a benzopyrene monoclonal antibody to specifically bind to benzo[a]pyrene in the sample. Vortex mix for 14 minutes and discard the supernatant. Add 1mL of methanol solution and vortex mix for 2 minutes to elute the benzo[a]pyrene bound to the beads. Dilute the beads 5-fold with a sustained-release solution (PBS containing 1.0% sucrose, 1.0% BSA, and 0.1% Tween-20) to prepare the sample for testing.

[0093] (2) Take 100 μL of each of the above-mentioned benzo[a]pyrene standard solutions and add them to the sample reaction bottle, mix them, insert the fluorescent test strips, react at 37°C for 6 minutes, absorb the residual liquid on the sample pad with absorbent paper, and immediately detect using a time-resolved fluorescence immunoassay (excitation wavelength: 365 nm, measurement wavelength: 615 nm). The fluorescence intensity values at the test line (T) and the control line (C) on each fluorescent test strip are obtained, and the ratio of the fluorescence intensity of the test line to the fluorescence intensity of the control line (T / C) of each fluorescent test strip is obtained. A standard curve is established with the standard concentration as the horizontal axis and the T / C value as the vertical axis. The quantitative detection limit is 0.5 ng / mL.

[0094] (3) Benzo[a]pyrene was added to blank corn oil samples at concentrations of 5 ng / mL, 10 ng / mL, and 50 μg / mL. The recovery rates of the additions were calculated using the standard curve and were between 90.3% and 110.2%.

[0095] Example 7: Application of the above-mentioned flow-delayed immunoassay time-resolved fluorescence rapid test kit in the detection of benzo[a]pyrene in actual corn oil samples:

[0096] Take 5 corn oil samples to be tested, and the pre-treatment and post-test steps are the same as in Example 2. Use a time-resolved fluorescence immunoassay to detect and obtain the ratio of the fluorescence intensity of the test line of each fluorescent test strip to the fluorescence intensity of the quality control line (T x / C), and then substitute it into the ratio of the fluorescence intensity of the fluorescent test strip detection line to the fluorescence intensity of the quality control (T x The correlation coefficient between the kit and the HPLC detection results reached 0.987 (R2), and the detection recovery rate was between 90.3% and 110.2%.

Claims

1. Benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit, characterized in that: The invention relates to a fluorescent test strip and a sample reaction cup containing a europium-labeled fluorescent probe, wherein the europium-labeled fluorescent probe is a freeze-dried europium-labeled anti-benzo[a]pyrene monoclonal antibody. The fluorescent test strip comprises a backing plate, one side of which is sequentially adhered with a water-absorbing pad, a detection pad, and a sample pad from top to bottom, with adjacent pads overlapping and connected at the joints. The detection pad uses a nitrocellulose membrane as a base and has a quality control line and a detection line arranged horizontally from top to bottom. The quality control line and the detection line are respectively coated with a goat anti-mouse secondary antibody and a benzo[a]pyrene detection antigen. The anti-benzo[a]pyrene monoclonal antibody is obtained by secretion of a hybridoma cell line BBBE1H1, which was deposited in the China Center for Type Culture Collection on April 3, 2018, at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: C201882.

2. The benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1, characterized in that: The europium labeling reagent is europium oxide latex fluorescent microspheres.

3. The benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1, characterized in that: The europium-labeled anti-benzo[a]pyrene monoclonal antibody lyophilized product is formed by coupling the surface-modified carboxyl group with the amino terminus of the anti-benzo[a]pyrene monoclonal antibody. The specific preparation method is as follows: the europium labeling reagent is added to a boric acid buffer solution and sonicated, and then an EDC solution is added, shaken and mixed, centrifuged, the supernatant is removed, the boric acid buffer solution is added for re-dissolution, shaken and mixed, the anti-benzo[a]pyrene monoclonal antibody is added, the reaction is shaken, the supernatant is removed by centrifugation, the solution is sealed, and the solution is packaged and freeze-dried.

4. The benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1, characterized in that: The benzo[a]pyrene detection antigen is BAP-OVA; the coating amount of the benzo[a]pyrene detection antigen required per centimeter of the detection line on the detection pad of the fluorescent test strip is 480 to 1000 ng; the coating amount of the goat anti-mouse secondary antibody required per centimeter of the quality control line is 100 to 900 ng; and the content of the fluorescent probe in the sample reaction cup is 0.2 to 1.0 μg.

5. The benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1, characterized in that: Also included are a sample release solution and a sample release solution pipette, wherein the sample release solution is: a PBS solution (v / v) containing 1.0% to 1.5% sucrose (w / w), 0.05% to 3.0% BSA (w / w), and 0.01% to 0.30% Tween-20; The sample pad in the test strip is 13mm-15mm long, the detection pad is 25mm-28mm long, and the absorbent pad is 17mm-20mm long. The distance between the detection line and the upper edge of the nitrocellulose membrane is 10-18mm, and the distance between the quality control line and the detection line is 2-6mm.

6. The benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1, characterized in that: The sample to be tested is edible vegetable oil. The pretreatment steps of the sample to be tested are: ultrasonic extraction of the edible vegetable oil with n-hexane, taking the supernatant, diluting it 8-12 times with water, then adding immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with sample release solution to obtain the supernatant, which is the sample solution to be tested.

7. The benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1, characterized in that: The fluorescent test strip is obtained by the following method: (1) Cut the absorbent paper into absorbent pads of the required size; (2) Preparation of test pad: The benzo[a]pyrene detection antigen is prepared into an antigen coating solution, which is then horizontally coated on a nitrocellulose membrane by streaking to obtain a test line; the goat anti-mouse secondary antibody is prepared into a concentration coating solution, which is then horizontally coated on a nitrocellulose membrane by streaking to obtain a quality control line; (3) Preparation of sample pad: Place the glass fiber membrane in the blocking solution and soak it completely. After taking it out, dry it at 37-40℃ overnight to obtain the required sample pad for later use. (4) Assembly of fluorescent test strips: The absorbent pad, the detection pad and the sample pad are sequentially pasted on one side of the liner, and the adjacent pads are overlapped and connected at the joints to obtain a fluorescent test strip.

8. Use of the benzo[a]pyrene flow lag immunoassay time-resolved fluorescence rapid test kit according to claim 1 in the detection of benzo[a]pyrene content: placing a sample reaction cup in a 37°C incubator, adding the sample solution to be tested into the sample reaction cup, pipetting and mixing, then inserting a fluorescent test strip, reacting for 6 to 10 minutes, and detecting using a time-resolved fluorescence meter to obtain the ratio of the fluorescence intensity value of the test line on the fluorescent test strip to the fluorescence intensity value of the quality control line; based on the previously obtained relationship curve between the ratio of the fluorescence intensity of the test line on the fluorescent test strip to the fluorescence intensity of the quality control line and the benzo[a]pyrene concentration, the benzo[a]pyrene content in the sample solution to be tested is obtained.

9. The use according to claim 8, characterized in that The sample to be tested is edible vegetable oil. The pretreatment steps of the sample to be tested are: ultrasonic extraction of the edible vegetable oil with n-hexane, taking the supernatant, diluting it 8-12 times with water, then adding immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with sample release solution to obtain the supernatant, which is the sample solution to be tested.

10. The use according to claim 8, characterized in that The relationship curve between the ratio of the fluorescence intensity of the test line of the fluorescent test strip to the fluorescence intensity of the quality control line and the concentration of benzo[a]pyrene is obtained by the following method: (1) preparing a series of benzo[a]pyrene standard solutions with a concentration gradient; (2) Add appropriate amounts of the above-mentioned benzo[a]pyrene standard solutions of each concentration into the sample reaction cup, mix well, insert the fluorescent test strip, react at 37°C for 6 minutes, and use a time-resolved fluorescence immunoassay to obtain the time-resolved fluorescence intensity values of the test line and the quality control line on each fluorescent test strip, thereby obtaining the ratio of the fluorescence intensity of the test line of each fluorescent test strip to the fluorescence intensity of the quality control line; (3) The relationship curve between the ratio of the fluorescence intensity of the test line of the fluorescent test strip to the fluorescence intensity of the quality control line and the concentration of benzo[a]pyrene was obtained by fitting.

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